There are a variety of conditions in humans which are characterized by a high level of bone resorption and by an abnormal balance between bone formation and bone resorption. Among the more common of these are osteoporosis, Paget's disease, and conditions related to the progress of benign and malignant tumors of the bone and metastatic cancers which have been transferred to bone cells from, for example, prostate or breast initial tumors. Other conditions which are associated with changes in collagen metabolism include osteomalacial diseases, rickets, abnormal growth in children, renal osteodystrophy, and a drug-induced osteopenia. Irregularities in bone metabolism are often side effects of thyroid treatments and thyroid conditions per se, such as primary hypothyroidism and thyrotoxicosis as well as Cushing's disease.
It has been recognized that disorders of bone resorption or other conditions characterized by an abnormal balance between bone formation and bone resorption can be detected by altered levels of pyridinium crosslinks in urine (Robins, 1982b; Macek; Black). The crosslinks take the form of compounds containing a central 3-hydroxy pyridinium ring in which the ring nitrogen is derived from the epsilon amino group of lysine or hydroxylysine (Fujimoto, 1978; Robins, 1982a; Gunja-Smith; Ogawa; Eyren).
The pyridinium crosslink compounds found in urine can be grouped into four general classes: (1) free, native crosslinks having a molecular weight of about 400 daltons (Fujimoto), (2) glycosylated crosslinks and crosslink peptide forms having a molecular weight of between about 550 and 1,000 daltons (Robins, 1983), (3) crosslink peptide forms having a molecular weight between 1,000 and 3,500 daltons (Robins, 1983, 1984, 1987; Henkel; Eyre), and (4) crosslink peptide forms having a molecular weight greater than 3,500 daltons. In normal adults, these forms account for about 38% (1), 40% (2), 15% (3), and 7% (4) of total urinary crosslinks. About 80% of the free crosslinks in normal adults is pyridinoline (or Pyd), derived from a hydroxylysine residue, and about 20%, deoxypyridinoline, or Dpd, derived from a lysine residue, and this ratio of Pyd/Dpd applies roughly to the other three classes of crosslinks in urine. The higher molecular weight crosslinks can be converted to free crosslinks by acid hydrolysis (Fujimoto, 1978).
Methods for measuring pyridinium crosslinks in urine have been proposed. One of these methods involves the measurement of total hydrolysed Pyd, i.e., Pyd produced by extensive hydrolysis of urinary crosslinks, by quantitating the hydrolysed Pyd peak separated by HPLC (Fujimoto, 1983). The relationship between total hydrolysed Pyd to age was determined by these workers as a ratio to total hydrolysed Pyd/creatinine, where creatinine level is used to normalize crosslink levels to urine concentration and skeletal mass. It was found that this ratio is high in the urine of children, and relatively constant throughout adulthood, increasing slightly in old age. The authors speculate that this may correspond to the loss of bone mass observed in old age.
Studies on the elevated levels of total crosslinks in hydrolyzed urine of patients with rheumatoid arthritis has been suggested as a method to diagnose this disease (Black). The levels of total hydrolyzed crosslinks for patients with rheumatoid arthritis (expressed as a ratio of total crosslinks measured by HPLC to creatinine) were elevated by a factor of 5 as compared to controls. However, only total hydrolysed Pyd, but not total hydrolysed Dpd, showed a measurable increase.
In a more extensive study using hydrolyzed urines, Seibel et al. showed significant increases in the excretion of bonespecific total hydrolysed Pyd crosslinks relative to controls in both rheumatoid and osteoarthritis, but the most marked increases for total hydrolysed Pyd were in patients with rheumatoid arthritis (Seibel).
More recently, the applicants have shown that a variety of bone collagen disorders, including osteoporosis, Paget's disease, osteoarthritis, hyperparathyroidism, and rheumatoid arthritis, can be detected on the basis of characteristic levels of urinary native Pyd or native Dpd. Levels of native Pyd or Dpd were measured by HPLC separation and quantitation of treated urine samples. The use of native crosslinks for detection of these bone disorders is advantageous in that the several-hour hydrolysis step needed to convert pyridinoline crosslinks to hydrolysed Pyd is avoided.
Assay methods, such as those just noted, which involve HPLC quantitation of crosslinks from hydrolysed samples, or crosslink subfractions from non-hydrolysed samples, require accurate calibration of the HPLC peak heights, in order to accurately quantitate each of the peaks. Ideally, an internal standard for use in an HPLC assay of urinary pyridinoline should (a) be recoverable in substantially the same yield as Pyd and Dpd during chromatographic fraction of a urine sample, (b) have similar spectroscopic (e.g. UV-visible absorbance and fluorescence) properties, and (c) be characterized by a retention time close to but distinct from the retention times of Pyd and DpD in chromatographic analysis (e.g., reversed phase C-18 HPLC).
Immunoassays have also been proposed for measuring urinary crosslinks. U.S. Pat. No. 4,973,666 discloses an assay for measuring bone resorption by detection in urine of specific pyridinium crosslinks, characterized by specific peptide extensions, associated with bone collagen. Two specific entities having peptide extensions presumed to be associated with bone collagen are described. These are obtained from the urine of patients suffering from Paget's disease, a disease known to involve high rates of bone formation and destruction. The assay relies on immunospecific binding of crosslink compounds containing the specific peptide fragment or extension with an antibody prepared against the crosslink peptide. It is not clear whether and how the concentration of crosslink peptide being assayed relates to total urinary crosslinks.
Robins has described a technique for measuring pyridinoline in urine by the use of an antibody specific to hydrolysed Pyd (Robins, 1986). The method has the limitation that the antibody was found to be specific for the hydrolyzed form of Pyd, requiring that the urine sample being tested first be treated under hydrolytic conditions. The hydrolytic treatment increases the time and expense of the assay, and precludes measurements on other native pyridinium crosslinks. More recently, the applicants have disclosed an enzyme immunoassay for detection of native Pyd in urine samples, for use in detecting a variety of bone collagen disorders.
The typical enzyme immunoassay format for detecting Pyd involves a solid-phase reagent containing surface-bound Pyd and a soluble anti-Pyd antibody, where sample Pyd competes with the surface-bound Pyd for binding to the soluble antibody. The extent of binding of antibody to the solid support thus provides a measure of Pyd concentration in the sample. In this format, it is desirable that the surfacebound Pyd resemble free Pyd in its antigenic characteristics, i.e., that its conjugation to the solid surface does not appreciably perturb its reaction affinity for the soluble antibody.
It is also desirable that the anti-Pyd antibody employed in the assay- whether a polyclonal or monoclonal reagent-has a high binding affinity for pyridinoline relative to more complex pyridinoline crosslinks, i.e., crosslinks containing additional glycosylation or peptide moieties, particularly when assaying non-hydrolysed samples. In preparing such an antibody reagent, the immunogen (which is typically a pyridinoline moiety conjugated to a carrier protein) should be relatively unobstructed at its pyridine ring and attached amino acid positions. At the same time, the immunogen should be structurally homogeneous, to reduce the range of antibodies which are produced in response to the imununogen, particularly when the immunogen is used for preparing a polyclonal antibody reagent.
4. Summary Of the Invention
The present invention includes a pyridinoline composition in which pyridinoline is derivatized specifically at its aliphatic hydroxyl group by a selected chemical group. For use as an internal standard, the derivatized pyridinoline is characterized by a retention time close, to but distinct from, the retention times of pyridinoline and deoxypyridinoline, as determined by reversed phase C-18 liquid chromatography. A preferred chemical group for this embodiment is an acetyl group.
In another embodiment, where the chemical group serves to anchor the pyridinoline to a solid support, the composition is useful in a solid support for an enzyme immunoassay.
In yet another embodiment, where the chemical group serves to anchor the pyridinoline to a carrier protein, the composition is useful for raising antibodies against pyridinium crosslink species.
In another aspect, the invention includes a method of derivatizing the aliphatic hydroxyl group of pyridinoline. In the method, anhydrous sample of pyridinoline is reacted with an acylating agent in anhydrous trifluoroacetic acid for a time sufficient to selectively derivatize the aliphatic hydroxyl group of the pyridinoline.
In one embodiment, the method is useful for preparing an acylated pyridinoline for use in quantitating pyridinoline in a urine sample.
In another embodiment, the method is useful for preparing a pyridinoline immunogen or a pyridinoline solid-surface reagent for immunoassay applications as above.
Also forming part of the invention is a method of quantitating pyridinoline crosslinks present in a urine sample. The method includes adding to the sample a known quantity of an O-acetylated pyridinoline, resolving the pyridinoline crosslinks and acetylated pyridinoline in the sample by reversed phase C-18 HPLC, and quantitating the separated crosslinks by use of the separated acetylated pyridinoline to calibrate the recovery of the crosslinks.
These and other objects and features of the invention will become more fully apparent when the following detailed description of the invention is read in conjunction with the accompanying drawings.